*2.3. Transfections, Selection, and Cell Cloning*

Electrotransfection of circular DNA was performed using a Bio-Rad Gene Pulser apparatus and electroporation conditions as described [39]. Briefly, promastigotes grown to mid-log phase were harvested by centrifugation (1251 *g*, 10 min, 4 ◦C), washed twice with ice-cold phosphate-buffered saline (PBS), once in pre-chilled electroporation buffer, and suspended in electroporation buffer at a density of 1 × 10<sup>8</sup> parasites/mL.

For the generation of double allele replacements and for the integration of linearised DNA constructs, cells were transfected following the Amaxa protocol as described previously [17,40]. Briefly, 1 × 10<sup>7</sup> promastigotes grown to mid- to late-log phase were harvested by centrifugation at 1251 *g* for 10 min (at RT), washed once with 1 × Tb-BSF electroporation buffer (90 mM NaHPO3, 5 mM KCl, 0.15 mM CaCl2, 50 mM HEPES, pH 7.3) [41] at RT, and suspended in 150 µL electroporation buffer per transfection. For gene editing, the cell suspension was mixed with the pooled unpurified PCR amplicons for the two single-guide RNA (sgRNA) templates and two donor DNAs (combined volume approximately 100 µL, heat-sterilised at 94 ◦C for 5 min before transfection) in a total volume of 250 µL. For integration of transgenes into the 18S SSU rRNA locus, cells were mixed with 2 µg of the *Swa*I-linearised DNA construct. Electroporation was performed in a 0.2 cm gap Gene Pulser electroporation cuvette (Bio-Rad, München, Germany) using one pulse with program X-001 in the Amaxa Nucleofector IIb device (Lonza, Basel, Switzerland). A mock transfection control without DNA was included to check the real transfection efficiency.

Following electroporation, cells were immediately transferred into 5 mL drug-free pre-warmed complete M199 medium in 25 cm<sup>2</sup> cell culture flasks. After parasite recovery at 25 ◦C for 16–20 h, the selection antibiotics were added at the indicated strain-specific concentrations. Nourseothricine (ClonNat, at 150 µg/mL for all parasite species; Werner BioAgents, Jena, Germany), hygromycin B (at 50 µg/mL for all parasite species; Roth, Karlsruhe, Germany), bleocin (at 5 µg/mL; Calbiochem, San Diego, CA, USA). Additionally, blasticidin (at 10 µg/mL for *L. donovani* and *L. major*; at 2.5 µg/mL for *L. braziliensis*; Roth, Karlsruhe, Germany) and puromycin (at 25 µg/mL for *L. donovani* and *L. major*; at 10 µg/mL for *L. braziliensis*; Sigma-Aldrich, München, Germany) were used to select for integration of the donor gene fragments. For *L. braziliensis*, double drug-resistant cell populations with the intended gene replacements were first selected at a lower selection pressure as indicated until they emerged in culture (about 2–3 weeks), followed by an increase in the selection pressure (at ~IC99.7:

5 µg/mL blasticidin; ~IC96: 20 µg/mL puromycin) to allow discrimination with the mock-transfected control cultures.

For cloning by limiting dilution, exponential log-phase cultures of the candidate *L. braziliensis HSP23-* and *HSP100*-null mutants were seeded in complete M199 medium at 0.5 cells per well in two 96-well microtitre plates, as described previously [39]. After 14 days, monitoring of wells for promastigote growth by light microscopy was started and continued until growth-positive wells were observed. The contents of positive wells were seeded into 2 ml complete M199 medium maintaining the drug pressure (blasticidin and puromycin at ~IC96–IC99.7) in 25 cm<sup>2</sup> cell culture flasks to expand the culture. Each population that emerged from an individual well was considered an individual clone.
